| Popis: |
Inside-out apical membrane vesicles were isolated from bovine tracheal epithelium. They were enriched 13- and 18-fold in two apical membrane markers, alkaline phosphatase and gamma-glutamyltransferase, respectively, and presented a low level of contamination by basolateral and intracellular membranes. These apical membrane vesicles of homogeneous inside-out orientation were used to measure 36Cl- influx. The 36Cl- influx was found to be (i) voltage-insensitive (ii) diphenylcarboxylic acid-insensitive, and (iii) from 55 to 100% activated by cAMP-dependent protein kinase according to initial rates and accumulation capacities. This rapid and ATP-dependent activation was associated with phosphorylation of a 170-180-kDa protein but was not observed with a nonhydrolyzable nucleotide like adenosine 5'-O-(3-thiotriphosphate). Immunodetection experiments showed that the mature form of bovine cystic fibrosis transmembrane conductance regulator (CFTR) was only present in the apical membranes. As compared with the previously described characteristics of CFTR, the 36Cl- uptakes detected here are the in vitro manifestation of the functional form of bovine CFTR located at the apical level in these tracheal epithelial cells. Inside-out apical membrane vesicles, with freely accessible cytoplasmic sides and functional CFTR, offer a new model system to study CFTR. |
